Utilization of the peroxidase-like activity of silver nanoparticles nanozyme on O-phenylenediamine/H2O2 system for fluorescence detection of mercury (II) ions

Polyvinylpyrrolidone stabilized silver nanoparticles (PV-AgNPs) were synthesized from AgNO3/trisodium citrate and with the assistance of microwave energy. The synthesized PV-AgNPs were found to own an actual peroxidase mimicking activity. This catalytic activity can oxidize the non-fluorescence reagent (o-phenylenediamine) to a high fluorescence reaction product (2,3-diaminophenazine). The reaction product exhibited a fluorescence emission at 563 nm upon the excitation at 420. Among many metals, only mercury (II) ions can inhibit the catalytic activity of PV-AgNPs nanozyme. Accordingly, the fluorescence intensity of the reaction product has been successfully quenched. This quenching effect in the fluorescence intensity was directly proportional to the concentration of mercury (II). Depending on this finding, a simple, cost-effective, and selective spectrofluorimetric approach has been designed for mercury (II) detection in water samples. The linear relationship between the inhibition in fluorescence intensity and mercury (II) concentration was found in 20–2000 nM with a detection limit of 8.9 nM.

www.nature.com/scientificreports/ are afforded with some features, including low cost, high stability, resistance to the high concentrations of the substrate, ease of storage process, and ease of synthesis [19][20][21] .
In general, nanoparticles of noble metals (such as gold, silver, platinum, and palladium) exhibit appealing physicochemical features that are dependent on their form and size 6,16 . For example, the peroxidase-like catalytic activity of gold nanoparticles has been utilized for the colorimetric sensing of mercury (II) and lead ions in water samples 22,23 . Furthermore, the catalytic activity of platinum nanoparticles has been utilized to detect mercury (II) ions in water samples 24,25 . Also, the catalytic activity of silver nanoparticles has been employed for visual colorimetric detection of protein and as a resonance Rayleigh scattering sensor for mercury (II) ions detection [26][27][28] . The catalytic activity of these nanomaterials depends on their sizes, known as the "size effect"; for instance, the great catalytic activity for gold nanoparticles can be observed with nano sizes smaller than 5.0 nm 16,29,30 . For that reason, a lot of efforts have been devoted to reducing the size of the synthesized nanoparticles 31 .
The reported studies prove that using polyvinylpyrrolidone surfactant to prepare Ag-NPs produces tiny nano sizes below 10 nm and stabilizes the formed nanoparticles for a long period 32,33 . Furthermore, microwave irradiation energy over conventional heating causes uniform and rapid heating to the solution. It thus produces homogeneous nucleation sites in the solution and growth conditions, causing monodispersed nanoparticles in a short time 34 . Moreover, microwave irradiation can be afforded good particle size distribution and smaller particle sizes to synthesize silver nanoparticles 35 .
Fluorescence-spectrometer is a highly sensitive analytical technique that usually offers great selectivity without losing precision [36][37][38] . Designing a fluorescent sensor for mercury (II) ions detection relying on the peroxidaselike property of silver nanoparticles has not been investigated yet. Therefore, this work aims to use the catalytic activity of the smaller size polyvinylpyrrolidone stabilized silver nanoparticles as a nanozyme for the fluorescence detection of mercury (II) ions.

Materials, instruments, and methods
Materials. O-Phenylenediamine, polyvinylpyrrolidone, and silver nitrate have been produced by Sigma-Aldrich Chemical Co (Steinheim, Germany).
Aluminum nitrate, barium chloride, cadmium chloride, chromium chloride, cobalt nitrate, calcium chloride, magnesium chloride, hydrogen peroxide, mercuric chloride, nickel nitrate, sodium chloride, potassium chloride, and zinc nitrate have been produced by El-Nasr chemical Co. (Cairo, Egypt). Trisodium citrate has been produced by Fisher Scientific Co. (Leicestershire, UK). Ultrapure water was utilized in all experimental steps.
Instruments. The fluorescence spectra were performed on an FS2 fluorescent spectrometer (Scinco, Korea).
The morphology of the prepared silver nanoparticles has been characterized by JSM 5400 LV SEM (JEOL, Tokyo, Japan). The nano size, poly dispersion index, and the quality of the prepared silver nanoparticles have been characterized by ZEN 1690 (Malvern Instruments, Malvern, UK). SM-2000MW microwave oven (Smart Co., China) was utilized for heating process.
Synthesis of silver nanoparticles using microwave energy. 0.2% w/v PVP solution, 10 mM trisodium citrate solution and 10 mM silver nitrate solution were simultaneously poured into a 250 mL flask in the ratio of 0.5:1:1 and mixed by magnetic stirring for 3 min. The flask has been heated for about 12 min at 90 °C by microwave irradiation. The formation of polyvinylpyrrolidone silver nanoparticles (PVP-AgNPs) can be evidenced by the transformation of the colorless solution to a yellowish-green colloidal state.

Detection of mercury (II) ion.
In a series of calibrated flasks (10 mL), suitable volumes of mercury (II) solutions (in the range of 100 nM to 20 µM) and 800 µL of PVP-AgNPs solution were poured, incubated for 2 min and followed by the addition of 800 µL from O-phenylenediamine (prepared by dissolving 0.108 g in 100 mL water) solution. Then, 400 µL of 3% w/v hydrogen peroxide solution was added into the content, and the content was vortexed for 1 min. After incubation for 15 min at room temperature, the volume has been completed to a 10-mL by deionized water. The blank solution has been simultaneously prepared by the same steps with omitting the addition of mercury (II) solution. The quenching in the fluorescence intensity of the blank solution upon the addition mercury (II) was measured at the emission of 563 nm, upon the excitation of 420 nm. The specificity of the suggested method has been checked by the addition of different metal ions solutions at the concentration of 10 µM instead of mercury (II) ion in the abovementioned procedures.

Detection of mercury (II) in different water samples using PVP-AgNPs. Tap water and bottled
water samples were collected from our laboratory and a local establishment. The collected samples were spiked with different known concentrations of mercury (II). Then after, the water samples were filtered utilizing a 0.45 μm syringe filter to discard any particulates matter. Finally, the abovementioned general analytical assay was followed.

Results and discussion
Characterization and peroxidase mimicking activity of PVP-AgNPs. The morphology and elemental characteristics [particle size, polydispersity index (PDI), size uniform] of PVP-AgNPs were examined using SEM device and zeta-sizer device, respectively. Figure 1 shows the SEM image of PVP-AgNPs, which refers to the spherical-rod-like shape of the synthesized nanomaterial. The measured size of the synthesized PVP-AgNPs was 5.5 nm with uniform size, good quality, and a low polydispersity index value of 0.440 Fig. 1A. The size of nanoparticles is the main factor responsible for their catalytic activity 39  www.nature.com/scientificreports/ formance of the silver nanoparticles is inversely proportional to the nanosize of their particles 40 . In the current study, the determined size of the prepared PVP-AgNPs is very small (5.5 nm), which refers to their superior catalytic activity. O-Phenylenediamine (OPD) is one of the typical substrates utilized to investigate the peroxidase-like activity of the nanoparticles 25,41 . OPD (colorless and non-fluorescent) has been oxidized by peroxidase mimicking activity of certain nanoparticles to 2,3-phenazinediamine (colored and fluorescent) 25 . Herein, the peroxidase mimicking activity of the prepared PVP-AgNPs has been examined by the fluorescence technique and the spectrophotometric technique using OPD/H 2 O 2 system. Practically, the catalytic activity of the prepared PVP-AgNPs has been spectrophotometrically confirmed by the appearance of characteristic absorbance peak at λ max = 420 nm, Fig. 2. Furthermore, it has been fluorometrically evidenced by the existence of a distinct fluorescence peak at λ emission = 563 upon λ excitation = 420, Fig. 2. Moreover, it was found that only the mixture of PVP-AgNPs/OPD/ H 2 O 2 exhibited this fluorescence behavior. In contrast, neither one of the PVP-AgNPs/OPD mixture, OPD/ H 2 O 2 mixture, and PVP-AgNPs/H 2 O 2 mixture has produced any fluorescence character at the same conditions. These absorbance peak and fluorescence emission peak values are matching to those of 2,3-phenazinediamine that were reported in studies of literature 25 . Fabrication and design of mercury (II) fluorescence detection sensing system. It is well known that the colored 2,3-phenazinediamine, which possesses a distinctive fluorescent behavior at λ emission = 563 nm/ λ excitation = 420 nm, is the oxidized product of o-phenylenediamine. Initially, the colorless solution changed from non-fluorescent to a bright fluorescence yellow solution when the PVP-AgNPs were added to the o-phenylenediamine/H 2 O 2 system. In this reaction, the prepared PVP-AgNPs owned peroxidase mimicking activity that can catalyze the oxidation of o-phenylenediamine with H 2 O 2 to yield 2,3-phenazinediamine as the main reaction product.
Mercury has a unique advantage over the rest of the elements through its ability to form an amalgam with certain elements such as gold, platinum, and silver 24,25,27,28,42,43 . Therefore, in this study, the formation of Ag-Hg amalgam produces an effective inhibition for the catalytic activity of PVP-AgNPs, combined with changing their surface properties. This catalytic inhibition effect of PVP-AgNPs prevents the transformation of the OPD/H 2 O 2 system to 2,3-phenazinediamine. Accordingly, a quenching action on the fluorescence intensity of the solution upon the addition of mercury (II) in comparison with the fluorescence intensity of blank sample (without Hg 2+ ), The linear relationship between the quenching of fluorescence emission at 563 nm and mercury (II) concentration was established in the ranging of 20 nM to 2 μM with the regression equation of y = 0.1352x + 13.51 , R 2 value of 0.998, and LOD value of 8.9 nM (S/N = 3, where N represents noise and S represents sensitivity). The statistical parameters for detecting Hg 2+ by the fluorescence methodology are presented in Table 1. Other common metals such as Al 3+ , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , K + , Mg 2+ , Ni 2+ , Na + , and Zn 2+ have been tested by the current fluorescent methodology to investigate the selectivity of the design sensing system. It was found that there is no obvious effect in the emission intensity of PVP-AgNPs/OPD/H 2 O 2 system have been detected upon adding of any metals from mentioned metals at higher concentration level (tenfold excess in compared to mercury (II)). In contrast, the emission intensity of PVP-AgNPs/OPD/H 2 O 2 has been significantly decreased in the presence of mercury (II) which refers to the good selectivity for the fabricated system (Fig. 5). The explanation for the perfect selectivity of the proposed method for mercury (II) may be attributed to the formation of Ag-Hg amalgam through a specific interaction between silver nanoparticles and mercury (II) ion 42 .  Table 2 refers to the good recovery and SD values for determining mercury (II) ions by the current method. These SD and recovery values evidenced the validity of accuracy and precision of the presented methodology for mercury (II) detection in tap water samples and bottled water samples.

Conclusions
In this study, the microwave irradiation energy was utilized to assist the synthesis of polyvinylpyrrolidone stabilized silver nanoparticles with very small nano sizes. The prepared silver nanoparticles showed distinctive peroxidase activity behavior. Based on the inhibition effect of mercuric (II) ions towards this peroxidase activity of the prepared silver nanoparticles, a fluorescent methodology with a highly sensitive and extremely selective